Protein Nanocage-Based Photo-Controlled Nitric Oxide Releasing

May 26, 2017 - Protein Nanocage-Based Photo-Controlled Nitric Oxide Releasing Platform ... *E-mail: [email protected]., *E-mail: [email protected]...
1 downloads 0 Views 3MB Size
Download PDF
Letter www.acsami.org

Protein Nanocage-Based Photo-Controlled Nitric Oxide Releasing Platform Xiao Li,† Yajie Zhang,† Jian Sun,† Weijian Chen,† Xuewei Wang,† Fenli Shao,‡ Yuyu Zhu,‡ Fude Feng,*,† and Yang Sun*,‡ †

Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, and ‡State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, School of Life Sciences, Nanjing University, Nanjing 210023, P. R. China S Supporting Information *

ABSTRACT: A photoactive NO releasing system was constructed by incorporation of NO-bound Fe−S clusters into horse spleen apoferritin cavities with high loading efficacy. The composites retained intact core−shell structure and indicated advantages such as enhanced stability, reduced cytotoxicity, efficient cellular uptake, and photocontrolled NO releasing property.

KEYWORDS: protein nanocage, nitric oxide, iron−sulfur cluster, light-controlled release, cell imaging

G

toxicity of metals when they are introduced to living objects, nanocarriers such as liposomes, proteins, and inorganic nanoparticles have shown improved performance with minimized cytotoxicity as well as targeted delivery capability.10−13 Iron−sulfur cluster molecules capable of liberating multiple NO molecules under light exposure have been well studied by Ford’s group,14 and recently were reported by Zhao and co-workers for codelivery with doxorubicin to HeLa cells using mesoporous-silica-coated upconversion nanoparticles as the photosensitizing platform.15 Different from most of reported nanostructures for drug delivery, natural apoferritin is unique for its reversible reassembling property and specific binding affinity toward transferrin receptor-overexpressing tumor cells.16 The large hollow cavity in a diameter of ∼8 nm allows for accumulation of guest molecules in quantity generally by a disassembly reassembly process, which makes it an ideal platform for drug delivery, cell imaging, nanoreactor, etc.17,18 The cavity of apoferritin originally acts as Fe reservoir to maintain iron homeostasis for normal physiological functions. The inner binding sites, natural or genetically modified with cysteines, for anchoring a variety of metals such as Fe and Cu are found useful to cage metal-coordinated complexes.19 Most recently, Fujita et al. employed mutant horse liver apoferritin with two

asotransmitters are cellular membrane permeable and play crucial roles in signal transduction processes.1 As the first found gaseous signal molecule, nitric oxide (NO) is now well-demonstrated to participate in neurotransmission, cardiovascular activity and immune system.2 The positive and negative effects of NO on disease process, such as tumor growth or suppression, are closely associated with the generation level of NO and cellular sensitivity. Increasing research has been focused on regulation of the NO level and its effect on tumor biology by activating or restraining NOS isoforms expressed in cells.3,4 Alternatively, the interference of NO-mediated signal transduction or pathological pathways in tumor progression and metastasis is achievable by delivery of exogenous NO as a therapeutic agent to living malignant cells.5 However, the therapeutic outcome of exogenous NO is limited by its short half-life and vulnerability to biological substances.6 This issue is usually resolved by the use of NO donors such as NONOates and S-nitrosothiol derivatives that are labile in physiological conditions to liberate gaseous NO. To satisfy the critical requirements for spatial and temporal control of NO release, photoactive NO donors are preferable by taking advantage of light as a trigger for controlled activation.7 For example, release of NO from S-nitrosothiol that was covalently connected to fluorescent carbon dot could be promoted by white LED light illumination.8 Metallic complexes with Ru, Mn, and Fe as centered metals and photolabile metal-NO coordination have been developed as photocontrolled NO releasing materials.9 Concerning the © 2017 American Chemical Society

Received: March 20, 2017 Accepted: May 26, 2017 Published: May 26, 2017 19519

DOI: 10.1021/acsami.7b03962 ACS Appl. Mater. Interfaces 2017, 9, 19519−19524

Letter

ACS Applied Materials & Interfaces

Figure 1. (a) Schematic representation of Apoferritin and chemical structures of RBS & RRS; (b) illustration of RBS incorporation and photoinduced NO release; (c) schematic representation of intracellular internalization of nanocomposites and photoinduced intracellular NO release.

Figure 2. (a) HR-TEM images of UA-stained apoferritin and RBS-NP, and nonstained RBS-NP (scale bar = 50 nm, inner 10 nm); (b) DLS analysis of apoferritin, RBS-NP, and light-treated RBS-NP; (c) CD spectra of apoferritin, RBS-NP, and light-treated RBS-NP.

cavity.21 This finding suggests accumulation of less hydrophilic metal complexes are more favorable. In the present work, we coupled horse spleen apoferritin with Roussin’s black salt (RBS), a multiple NO-bound iron−sulfur cluster (structurally shown in Figure 1a),22 to obtain protein composites with the following merits: enhanced loading capacity without the need of inner surface engineering, photocontrolled NO release (Figure 1b), high NO supply level, retention of cytotoxic iron ions after Fe-NO dissociation, and promoted intracellular internalization (Figure 1c). The factors such as availability of iron binding sites and hydrophobic nature of iron−sulfur-based NO-releasing agents are expected to contribute to the enhanced loading capacity. The acid-unstable Fe−S clusters would be decomposed if pH-induced disassembly reassembly of apoferritin was applied

cysteines per subunit in close proximity to accumulate MnCO complexes up to 44 moieties per cage, and applied the photoactive CO-releasing protein cage for activation of NF-κB and TNF-α in mammalian cells under light after receptormediated cell internalization.20 Obviously, the use of genetic engineering technique may provide precise binding sites for selected metals particularly for those with poor binding affinity toward nonengineered apoferritin. Although the existence of metal binding sites is regarded as a necessity for anchoring of metal complexes as many as possible, the role of ligands is usually overlooked. In the previous report, we demonstrated that incorporation of a series of cationic Ru complexes bearing various ligands is strongly dependent on their hydrophobicity to fit in well in the hydrophobic protein 19520

DOI: 10.1021/acsami.7b03962 ACS Appl. Mater. Interfaces 2017, 9, 19519−19524

Letter

ACS Applied Materials & Interfaces

Figure 3. (a) Absorption spectra of RBS-NP before and after light exposure; (b) plots of photogenerated NO concentration from RBS and RBS-NP as a function of light irradiation time.

incorporation process ensures to maintain the α-helixes and βsheets of protein shell in the original state. The resulted nanocomposites treated with or without light exposure were stable in PBS buffer during a 2-month storage without precipitates. However, serious precipitation occurred in the control solution containing RBS alone (Figure S2a), likely due to the hydrolysis of RBS, which is known to be moisture sensitive.25 The bands of RBS-NP at 320−500 nm ranges after a 2 month storage remained visible in UV−vis absorption spectrum (Figure S2b), implying the Fe-NO bonding survived in the cage. It seems apoferritin protects Fe−S clusters from water contact and prevents Fe leakage from cage interior. To further evaluate the stability of RBS with or without caging treatment, the level of NO arising from automatic Fe-NO dissociation was determined in RBS and RBS-NP solutions containing 1.0 μg/mL iron at one-day intervals during dark storage over a period of 7 days. As shown in Figure S2c, the NO concentration in RBS solution gradually increased to 13 μM, much higher than 4.4 μM NO in the RBS-NP solution. In consideration of that the potential coordination interaction between Fe and amino acid residues may lead to weakened Fe-NO bonding, we checked Fe−NO bonding by UV−vis absorption spectroscopy. The absorption bands of RBS at 320 to 500 nm ranges in PBS (pH 7.4) disappeared after complete NO release by white LED irradiation (12 mW/cm2, 60 min) (Figure S3). The light irradiation caused the same changes of absorption spectra of RBS-NP, which suggests that Fe−NO bonding was uninterrupted by the incorporation process and remained photoactive (Figure 3a). The travel distance of NO in aqueous media is usually no longer than 500 μm,2 allowing NO to diffuse from protein cage into the outer solution upon light-induced Fe-NO dissociation and be quantitatively detected by Griess reaction method. As shown in Figure 3b, linear irradiation time-dependent NO accumulation was observed in a broad scale of NO generation at a steady rate of 4.6 and 3.3 μM/min for RBS and RBS-NP, respectively. It was noted that NO was readily detected in RBS solution even though carefully protected from light, due to the limited stability of RBS in aqueous media. The initial NO level was evidently lowered in RBS-NP solution, indicating improved stability of RBS by being caged in the hydrophobic environment. Concerning the potential reactivity of photogenerated NO toward protein structure, we carried out CD measurements which exhibited undetectable spectral changes before and after treatment with light exposure (Figure 2c), in addition to DLS analysis, which provided average hydrodynamic diameters of RBS-NPs unaffected by light treatment (Figure 2b). These results support that RBS-NP could be utilized as a photocontrolled platform for NO release without damage of cage structure.

for RBS incorporation. Thus, the loading of RBS into apoferritin was carried out in 10% acetonitrile-PBS buffer at a constant pH 7.4 (Figure 1b), at a molar ratio between Fe atom and protein of 500:1. After dialysis against 10% acetonitrile-PBS and then PBS, and subsequent purification on a PD-10 column, a clear brown solution of RBS-encapsulated protein composites (RBS-NP) was achieved. The protein concentration was examined by standard BCA assay (see Figure S1 for standard curves). The Fe loading was estimated to be 213 and 201 atoms per protein by the KMnO4 oxidation method and ICP-OES analysis, respectively. The similar values indicated reliability of both methods for quantification of Fe content in protein. If the loading molar ratio was lowered to 420:1, the Fe loading was slightly decreased to 210 atoms per protein as estimated by the KMnO4 oxidation method. Therefore, apoferritin is able to accommodate the 4Fe-3S sulfur cluster up to ∼53 moieties, in much greater number than the other known iron−sulfur proteins. RRS-NPs formed by complexation of apoferritin with a more photoactive Fe−S cluster, Roussin’s red salt (RRS, structurally shown in Figure 1a), had slightly improved stability, but not suitable for further investigation because of the rapid spontaneous loss of bounded NO even in dark. The high RBS loading number could not be well explained by the potential Fe binding in the channels. Niemeyer et al. identified the Fe binding sites in 2-fold channels that could only accommodate a total number of 12 ferrocenes, but additional possible binding sites were not detected by X-ray analysis of crystal structure.23 HR-TEM is a very powerful tool to analyze metal nanoparticles, and may provide more evidence for the location of metal complexes. The intact structure of RBS-NP was confirmed by HR-TEM using uranyl acetate as negative staining agent, in a similar pattern with apoferritin that showed transparent shells and dense cores stained by uranyl acetate (Figure 2a). When imaged by nonstained HR-TEM, RBS-NPs were visualized as punctas in the size comparable to protein cavity (Figure 2a). These data evidently indicate that RBS complexes were largely accumulated in the cage interior, likely due to the interactions of Fe−S cluster with histidine, arginine and tryptophan and other amino acid residues as well as the hydrophobic environment of cage interior. The average hydrodynamic diameter of RBS-NPs was slightly greater than apoferritin (12.9 ± 2.6 nm versus 10.0 ± 0.9 nm, shown in Figure 2b), as determined by dynamic light scattering (DLS) measurements. This phenomenon was common to encapsulation with high loading of guest molecules.21,24 To check the changes of protein’s secondary and tertiary structures before and after RBS encapsulation, we performed circular dichroism (CD) spectrometry. As shown in Figure 2c, the positive peaks at 193 nm and negative peaks at 208 and 222 nm in CD spectra were constant, which means the disassembly free 19521

DOI: 10.1021/acsami.7b03962 ACS Appl. Mater. Interfaces 2017, 9, 19519−19524

Letter

ACS Applied Materials & Interfaces

Figure 4. (a) Fluorescent images of RBS or RBS-NP-treated HeLa cells before and after light exposure using DCF-FM DA as a NO probe; (b) MTT analysis of HeLa cells treated by RBS and RBS-NP at different Fe concentrations; (c) MTT analysis of apoferritin, or RBS, or RBS-NP-treated HeLa cells with light exposure of varying irradiation time.

is cell type-dependent mechanism in response to oxidative stress. The cytotoxicity of RBS and RBS-NP in dark was characterized with IC50, defined as that Fe concentration required to inhibit 50% cell growth, by MTT assay. HeLa and MCF cells were treated with RBS or RBS-NP at varied Fe concentrations (0.5, 1, 2, 3, 4, 6, 8, and 10 μg/mL) for 48 h before MTT assay. The IC50 values in HeLa cells were obtained as 3.7 and 5.4 μg/mL Fe for RBS and RBS-NP, respectively (Figure 4b). The reduced cytotoxicity of RBS-NP in dark relative to RBS was also observed with MCF-7 cells (Figure S6). For normal cell, RBS and RBS-NP exhibited similar iron concentration-dependent viabilities on human hepatocyte QSG cells, with IC50 values increased to ∼10 μg/mL (Figure S7). The phototoxicity mediated by NO releasing was investigated on HeLa and MCF-7 cells. The cells were treated with RBS or RBS-NP at a Fe dose of 1.0 μg/mL for 16 h, then subjected to white LED light exposure (12 mW/cm2) for varying minutes (0, 5, 10, 15, 20, 25, and 30 min), followed by incubation for additional 24 h. Treatment of cells with PBS or apoferritin was used as control. The cell viabilities were determined by MTT assay. As shown in Figure 4c, treatment of HeLa cells with apoferritin, or light alone, or RBS in dark, or RBS-NP in dark did not affect cell viability. However, phototoxicity occurred in an irradiation time-dependent manner, or in other words, showing a dependence on photogenerated NO level on a basis that other photoproducts were not responsible for cell death.28 In contrast, RBS- or RBSNP-treated MCF-7 cells exhibited higher tolerance to light exposure (Figure S8), due to the aforementioned suppression of intracellular oxidative stress in MCF-7 cells. The preliminary

Because the Fe−S clusters were caged rather than absorbed at the apoferritin outer shell, the cellular internalization of apoferritin materials would not be affected by Fe−S clusters (Figure 1c). Two tumor cell lines, HeLa and MCF-7, were tested for in vitro investigation of RBS-NP photoactivity and toxicity in comparison with RBS. The cells were incubated with RBS-NP for 16 h, washed with PBS to remove noninternalized particles and then treated with a commercial NO probe, 3amino, 4-aminomethyl-2′,7′-difluorescein diacetate (DCF-FM DA),26,27 for 10 min before white LED light exposure (12 mW/ cm2) for 30 min. Under fluorescent microscope, the bright green image of RBS-NP-treated HeLa cells reflected efficient cellular uptake as well as light-induced NO releasing (Figure 4a). In contrast, the control group of RBS-NP without light treatment was imaged at low background signal. The cells treated by RBS gave similar imaging results, as Fe−S clusters were permeable to cell membranes and the Fe−NO bonding could survive in the cells. However, MCF-7 cells treated by RBS or RBS-NP and light were imaged with significantly decreased emission intensity (Figure S4), presumably owing to the shortened NO lifetime by the presence of intracellular scavenging reactions. To investigate if the cell response was linked to intracellular oxidative stress level, we imaged H2O2-treated HeLa and MCF7 cells with or without light exposure using a fluorescent reaction oxygen species (ROS) probe, DCFH-DA. As shown in Figure S5, light exposure alone did not affect ROS level. However, MCF-7 cells exhibited suppression of ROS stress upon the presence of H2O2, different from HeLa cells, which showed elevated intracellular ROS level. Hence, it seems there 19522

DOI: 10.1021/acsami.7b03962 ACS Appl. Mater. Interfaces 2017, 9, 19519−19524

Letter

ACS Applied Materials & Interfaces results reveal the correlation between the light flux and cell killing efficacy. The cells took up RBS and RBS-NP in different pathways. The small-sized hydrophobic RBS was cell membrane permeable, while RBS-NP could only be internalized by receptors-mediated endocytosis, which led to difference in iron uptake rate. The HeLa and MCF cells were treated by RBS and RBS-NP at the same initial iron concentration (3 μg/mL) in serum-free medium away from light for 1 or 2 h. The amounts of iron remaining in the medium were quantified by the KMnO4-oxidation method, to achieve the iron uptakes by subtraction of the determined iron amounts from the initial iron loading. As shown in Figure S9, for HeLa cells, RBS-NP treatment led to 14 and 5.3% more iron uptakes than RBS at 1 and 2 h post incubation, respectively; in a similar trend, for MCF-7 cells, RBS-NP treatment gave rise to 18 and 12% more iron uptakes than RBS during a period of 1 and 2 h, respectively. Consequently, RBS uptakes can be enhanced by the caging strategy. In summary, we incorporated photoactive Fe−S clusters into the cage interior of apoferritin at pH 7.4, avoiding traditional protein disassembly reassembly process. The bulky RBS complex was encapsulated at a loading efficiency of 42%, with a loading number up to 53 moieties per cage. The moisturesensitive RBS was significantly stabilized by the hydrophobic protein cage and the nanosized composites could be stored in PBS buffer with a shelf life of at least two months. The efficient cellular uptake and photocontrolled NO releasing property of RBS composites allow for cell-type-dependent medicinal applications in NO-based therapeutics.

■

(2) Beckman, J.; Koppenol, W. H. Nitric Oxide, Superoxide, and Peroxynitrite: The Good, the Bad, and the Ugly. Am. J. Physiol.: Cell Physiol. 1996, 271, C1424−C1437. (3) McCarthy, H. O.; Coulter, J. A.; Robson, T.; Hirst, D. G. Gene Therapy via Inducible Nitric Oxide Synthase: A Tool for the Treatment of a Diverse Range of Pathological Conditions. J. Pharm. Pharmacol. 2008, 60, 999−1017. (4) De la Rosa, A. J.; Rodriguez-Hernandez, A.; Gonzalez, R.; Romero-Brufau, S.; Navarro-Villaran, E.; Barrera-Pulido, L.; Pereira, S.; Marin, L. M.; Lopez-Bernal, F.; Alamo, J. M.; Gomez-Bravo, M. A.; Padillo, F. J.; Muntane, J. Antitumoral Gene-Based Strategy Involving Nitric Oxide Synthase Type III Overexpression in Hepatocellular Carcinoma. Gene Ther. 2016, 23, 67−77. (5) Sortino, S. Light-Controlled Nitric Oxide Delivering Molecular Assemblies. Chem. Soc. Rev. 2010, 39, 2903−2913. (6) Kumar, S.; Singh, R. K.; Bhardwaj, T. R. Therapeutic Role of Nitric Oxide as Emerging Molecule. Biomed. Pharmacother. 2017, 85, 182−201. (7) Fraix, A.; Marino, N.; Sortino, S. Phototherapeutic Release of Nitric Oxide with Engineered Nanoconstructs. Top. Curr. Chem. 2016, 370, 225−257. (8) Xu, J.; Zeng, F.; Wu, H.; Hu, C.; Yu, C.; Wu, S. Preparation of a Mitochondria-Targeted and NO-Releasing Nanoplatform and its Enhanced Pro-Apoptotic Effect on Cancer Cells. Small 2014, 10, 3750−3760. (9) Agustin, E. P.; Dayana, A. M.; Alexis, D. O.; Peter, C. F. In Luminescent and Phtoactive Transition Metal Complexes as Biomolecular Probes and Cellular Reagents; Kenneth Kam, W. L., Eds.; Springer: Berlin, 2015; Chapter 1, pp 1−45. (10) Hrabie, J. A.; Saavedra, J. E.; Roller, P. P.; Southan, G. J.; Keefer, L. K. Conversion of Proteins to Diazeniumdiolate-Based Nitric Oxide Donors. Bioconjugate Chem. 1999, 10, 838−842. (11) Zhang, J.; Yuan, G.; Feng, S.; Gong, Z.; Zhang, Y. Interaction Characteristics with Bovine Serum Albumin and Retarded Nitric Oxide Release of ZCVI4-2, a New Nitric Oxide-Releasing Derivative of Oleanolic Acid. Chem. Pharm. Bull. 2011, 59, 734−741. (12) Xiang, H. J.; An, L.; Tang, W. W.; Yang, S. P.; Liu, J. G. PhotoControlled Targeted Intracellular Delivery of Both Nitric Oxide and Singlet Oxygen Using a Fluorescence-Trackable Ruthenium Nitrosyl Functional Nanoplatform. Chem. Commun. 2015, 51, 2555−2558. (13) Nakanishi, K.; Koshiyama, T.; Iba, S.; Ohba, M. Lipophilic Ruthenium Salen Complexes: Incorporation into Liposome Bilayers and Photoinduced Release of Nitric Oxide. Dalton Trans. 2015, 44, 14200−14203. (14) Ford, P. C. Photochemical Delivery of Nitric Oxide. Nitric Oxide 2013, 34, 56−64. (15) Zhang, X.; Tian, G.; Yin, W.; Wang, L.; Zheng, X.; Yan, L.; Li, J.; Su, H.; Chen, C.; Gu, Z.; Zhao, Y. Controllable Generation of Nitric Oxide by Near-Infrared-Sensitized Upconversion Nanoparticles for Tumor Therapy. Adv. Funct. Mater. 2015, 25, 3049−3056. (16) Blight, G. D.; Morgan, E. H. Transferrin and Ferritin Endocytosis and Recycling in Guinea-Pig Reticulocytes. Biochim. Biophys. Acta, Mol. Cell Res. 1987, 929, 18−24. (17) Zhen, Z.; Tang, W.; Todd, T.; Xie, J. Ferritins as Nanoplatforms for Imaging and Drug Delivery. Expert Opin. Drug Delivery 2014, 11, 1913−1922. (18) Molino, N. M.; Wang, S.-W. Caged Protein Nanoparticles for Drug Delivery. Curr. Opin. Biotechnol. 2014, 28, 75−82. (19) Jutz, G.; van Rijn, P.; Santos Miranda, B.; Böker, A. Ferritin: A Versatile Building Block for Bionanotechnology. Chem. Rev. 2015, 115, 1653−1701. (20) Fujita, K.; Tanaka, Y.; Abe, S.; Ueno, T. A Photoactive CarbonMonoxide-Releasing Protein Cage for Dose-Regulated Delivery in Living Cells. Angew. Chem., Int. Ed. 2016, 55, 1056−1060. (21) Li, X.; Zhang, Y.; Chen, H.; Sun, J.; Feng, F. Protein Nanocages for Delivery and Release of Luminescent Ruthenium(II) Polypyridyl Complexes. ACS Appl. Mater. Interfaces 2016, 8, 22756−22761.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b03962.

■

Experimental details and figures (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Fude Feng: 0000-0002-5348-5959 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We’re grateful to Prof. Wei Wang (Nanjing University) for help with DLS measurements. We thank the National Basic Research Program of China (2015CB856300), 1000 Young Talent Program, Interdisciplinary Training for Graduate Students of Nanjing University (2016CL11), National Natural Science Foundation of China (81422050), Collaborative Innovation Center of Chemistry for Life Sciences, and the Program for Changjiang Scholars and Innovative Research Team in University for financial support.

■

REFERENCES

(1) Wang, R. Gasotransmitters: Growing Pains and Joys. Trends Biochem. Sci. 2014, 39, 227−232. 19523

DOI: 10.1021/acsami.7b03962 ACS Appl. Mater. Interfaces 2017, 9, 19519−19524

Letter

ACS Applied Materials & Interfaces (22) Bourassa, J.; DeGraff, W.; Kudo, S.; Wink, D. A.; Mitchell, J. B.; Ford, P. C. Photochemistry of Roussin’s Red Salt, Na2[Fe2S2(NO)4], and of Roussin’s Black Salt. J. Am. Chem. Soc. 1997, 119, 2853−2860. (23) Niemeyer, J.; Abe, S.; Hikage, T.; Ueno, T.; Erker, G.; Watanabe, Y. Noncovalent Insertion of Ferrocenes into the Protein Shell of Apo-Ferritin. Chem. Commun. 2008, 48, 6519−6521. (24) Zhen, Z.; Tang, W.; Guo, C.; Chen, H.; Lin, X.; Liu, G.; Fei, B.; Chen, X.; Xu, B.; Xie, J. Ferritin Nanocages to Encapsulate and Deliver Photosensitizers for Efficient Photodynamic Therapy against Cancer. ACS Nano 2013, 7, 6988−6996. (25) Seyferth, D.; Gallagher, M. K.; Cowie, M. The Bridging Sulfide Anion Reactivity of Roussin’s Red Salt. Organometallics 1986, 5, 539− 548. (26) Wang, Z.; Lu, Y.; Qin, K.; Wu, Y.; Tian, Y.; Wang, J.; Zhang, J.; Hou, J.; Cui, Y.; Wang, K.; Shen, J.; Xu, Q.; Kong, D.; Zhao, Q. Enzyme-Functionalized Vascular Grafts Catalyze in-Situ Release of Nitric Oxide from Exogenous NO Prodrug. J. Controlled Release 2015, 210, 179−188. (27) Wang, M.; Chen, M.; Ding, Y.; Zhu, Z.; Zhang, Y.; Wei, P.; Wang, J.; Qiao, Y.; Li, L.; Li, Y.; Wen, A. Pretreatment with βBoswellic Acid Improves Blood Stasis Induced Endothelial Dysfunction: Role of eNOS Activation. Sci. Rep. 2015, 5, 15357. (28) Janczyk, A.; Wolnicka-Glubisz, A.; Chmura, A.; Elas, M.; Matuszak, Z.; Stochel, G.; Urbanska, K. NO-Dependent Phototoxicity of Roussin’s Black Salt against Cancer Cells. Nitric Oxide 2004, 10, 42−50.

19524

DOI: 10.1021/acsami.7b03962 ACS Appl. Mater. Interfaces 2017, 9, 19519−19524